The human vocal folds (VFs, sometimes referred to as vocal cords or vocal chords), which are essential to the production of speech (phonation), are made up of a pair of structures that are stretched horizontally across the top of the trachea, within the larynx. The VF inner region of is made up of the vocalis muscle, which has both passive and active mechanical properties. Passively, the vocalis muscle has a relatively stiff consistency (sometimes compared to the consistency of a stiff rubber band), while, as a muscle, its active contractile properties help control its precise location and level of stiffness.
The vocalis muscle is covered by two mechanically de-coupled regions, each containing multiple layers. The region making up the outer covering of the vocal fold is known as the vocal fold mucosa (VFM). The vocal fold mucosa includes the outermost squamous epithelium layer and a mucosal lamina propria layer directly underneath the squamous epithelium. The squamous epithelium, which is composed primarily of stratified vocal fold epithelial cells (VFEs), serves as an initial boundary of protection for the underlying tissue and helps regulate vocal fold hydration. The underlying mucosal lamina propria, which is composed primarily of loose fibrous and elastic components in a vascularized matrix that is populated by vocal fold fibroblasts (VFEs), provides a pliant cushion having the mechanical properties needed for the vocal folds to vibrate in a manner that facilitates phonation.
The vocal folds also include a third region interposed between the inner vocalis muscle and the outer mucosa: the vocal ligament. The vocal ligament, which includes two non-mucosal lamina propria layers (the intermediate lamina propria and the deep lamina propria), is composed primarily of elastic and collagenous fibers, which provide this intermediate region with its elastic mechanical integrity and durability. See, e.g., Hirano M. Structure and vibratory behavior of the vocal fold, in Sawashima M, Cooper F (eds) (1977), Dynamic aspects of speech production, University of Tokyo, Tokyo, Japan: 13-30.
When inhaling, the vocal folds are separated, to facilitate the free flow of air through the trachea. When holding one's breath, the vocal folds tighten and come together, completely shutting off the free flow of air through the trachea. During phonation, the vocal folds are in an intermediate position, and the controlled passage of air from the lungs through the trachea causes the mucosa of adjoining vocal folds in contact with each other to vibrate at a high frequency. This transduction of energy from air flow from the lungs into high frequency vocal fold vibration in turn results in airborne sound waves that can be heard as speech. See, e.g., Matsushita H. The vibratory mode of the vocal folds in the excised larynx, Folia Phoniatr (Basel) 27 (1975): 7-18. Accordingly, phonation requires that both vocal fold mucosae are biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration, and physiologically capable of maintaining a barrier against the airway lumen.
Voice impairment (dysphonia) affects an estimated 20 million people in the United States, resulting in reduced general and disease-specific quality of life1, reduced occupational performance and attendance2,3, and direct health care costs exceeding $11 billion per year4,5. Between 60 and 80% of voice complaints in the treatment-seeking population involve changes to the vocal fold (VF) mucosa6; severe mucosal impairment or loss due to trauma, disease, or disease resection often culminates in fibrosis and deterioration of VF vibratory capacity for voice7.
Patients with significant VF mucosal damage have limited treatment options. Medialization of the impaired VF, achieved by delivering an implant or injectate to the paraglottic space8-10, can improve VF closure and therefore voice, but does not address fibrotic changes within the extracellular matrix (ECM). Superficial injection of regenerative biomaterials offers an alternative means to improve VF viscoelasticity and vibratory function11,12; however, most biomaterials are not specifically engineered for the VF biomechanical environment, have limited residence time, and are not suited for large deficits involving extensive tissue loss. Creation of an organotypic bioengineered VF mucosa could theoretically bypass these challenges by providing on-demand tissue for transplantation that is both biomechanically appropriate for use as a dynamic sound source for voice production and capable of maintaining barrier function at the boundary of the upper and lower airways.
Tissue engineering of partial and complete VF mucosae has been attempted using decellularized ECM-based13 and collagen14,15 and fibril16,17 gel-based scaffolds, seeded with embryonic stem cell derivatives15, adult stem cells16,17, and terminally differentiated cells13,14,18. These organotypic culture approaches have generated engineered mucosae with desirable histologic features; however, to date, there is no benchmark culture system based solely on human-sourced VF cells against which stem cell-based approaches can be evaluated, no direct comparisons showing equivalency with native human VF mucosa, and most importantly, limited progress towards the restoration of physiologic function17. Significant advances have been hampered by the near-unavailability of disease-free primary human VF mucosal cells19 and limited attention to the intricate protein- and anatomic substructure-level complexity that characterizes mucosal morphogenesis.
Accordingly, there is a need for improved non-immunogenic transplantable engineered mucosae that are biomechanically capable of aerodynamic-to-acoustic energy transfer and high-frequency vibration, and physiologically capable of maintaining a barrier against the airway lumen.